Method for producing biodiesel

ABSTRACT

The present disclosure is directed to the method for producing the biodiesel comprising steps of providing a biomass having an oil, obtaining a crude mixture of the biomass, and then performing a transesterification in which the crude mixture is used as a reacting material thereof to produce the biodiesel.

CROSS-REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY

The application claims the benefit of Taiwan Patent Application No. 101114435, filed on Apr. 23, 2012, in the Taiwan Intellectual Property Office, the disclosures of which are incorporated herein in their entirety by reference.

TECHNICAL FIELD

The present disclosure is directed to a method of producing the biodiesel. Specifically, the present disclosure relates to a method for producing the biodiesel by disrupting a biomass having an oil to obtain a crude mixture of the biomass and then performing a transesterification in which the crude mixture is directly used as a reacting material of the transesterification to produce the biodiesel.

BACKGROUND

Taiwan Patent Application No. 097135046 discloses a method for producing the biodiesel comprising following main steps of: mixing plant oil, animal oil and methanol to obtain a mixture, making the mixture reveal an uniformly ultrafine molecules state by ultrasonication, and then performing the transesterifications for the mixture many times via slow mixer and high-speed swirler to generate glycerol and methanol. The mixture having the glycerol and methanol further mixes with water, centrifuges the mixture to separate impurities, water and highly pure methanol from the mixture and then distils the final mixture to obtain the biodiesel.

Taiwan Patent Application No. 099115609 discloses an integrated technique of algae biodiesel and recycling of algae by fermentation, wherein microalgae grown in the photoreactor put in the oil-extracted device for the extraction of oil, and the oil contained in the microalgae is obtained and can be further transforming into the biodiesel.

Besides, it is a common technique to dry the microalgae in advance and then extract the oil of the dried microalgae for the preparation of biodiesel.

The above-mentioned techniques can only use the extracted oil of biomass, such as microalgae, or the plant and/or animal oils to be the raw materials for the preparation of biodiesel. However, the extracting procedures of microalgae oil in those techniques are complicated so as to not only make the costs thereof are high but also cause the extracted microalgae oil to be easily lost during these extracting procedures. Besides, the plant and/or animal oils are known as expensive if they are really used to be the industrial raw materials. Accordingly, those techniques are not economically effective.

Employing experiments and researches full-heartily and persistently, the applicant finally conceived the method for producing biodiesel.

SUMMARY

The present method produces the biodiesel by disrupting a biomass having an oil to obtain a crude mixture thereof and then performing a transesterification in which the crude mixture is directly used as a reacting material of the transesterification to produce the biodiesel.

On one aspect, the present disclosure provides a method for producing a biodiesel, comprising steps of providing a microalga; disrupting the microalga to obtain a disrupted microalga; and mixing the disrupted microalga directly with a catalyst, an alcohol and a co-solvent to produce the biodiesel.

On one aspect, the present disclosure provides a method for producing a biodiesel, comprising steps of providing a microalga; disrupting the microalgae to obtain a disrupted microalga; and performing a transesterification in which the disrupted microalga is directly applied to be a reacting material thereof to produce the biodiesel.

On one aspect, the present disclosure provides a method for producing a biodiesel, comprising steps of providing a biomass having an oil; obtaining a crude mixture of the biomass; and performing a transesterification in which the crude mixture is used as a reacting material thereof to produce the biodiesel.

The “crude mixture” in the present disclosure means a mixture obtained by breaking/disrupting cell of object, e.g. a biomass, without removing any ingredient therefrom.

The “disrupted microalga” in the present disclosure means a microalga mixture in which the microalga is broken/disrupted and no any ingredient is removed therefrom. That is, the “disrupted microalga” has all the ingredients, e.g. the whole ingredients, of the microalga.

The term “breaking” or “disrupting” refers to a process of disrupting the cell membrane and/or cell wall to make the contents in cell be released to form a cell lysate mixture.

The term “co-solvent” refers to a solvent in which the oil can be solved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of the present method of producing the biodiesel.

DETAILED DESCRIPTION

The present disclosure can be fully understood and accomplish by the skilled person according to the following embodiments. However, the practice of the present method is not limited into following embodiments.

Please refer to FIG. 1 which is a flow chart showing the present method of producing the biodiesel. As shown in the flow chart, the microalgae for producing the biodiesel can be provided from dry microalgae (produced by drying the naturally wet microalgae) mixed with a liquid (step 11) and/or wet microalgae (i.e. microalgae with liquid, mainly water) (step 12), wherein the liquid mixed with the dry microalgae can be water and/or other appropriate ones. Besides, either the dry microalgae mixed with liquid or the wet microalgae are able to adjust the volume of liquid, e.g. adding additional water into the dry or wet microalgae or removing the original water from the wet microalgae, thereof for the further processes, if necessary.

Then the dry microalgae mixed with liquid or the wet microalgae are processed by the disrupting step (step 13). After the disrupting step, the cell wall and cell membrane of microalgae are disrupted and therefore cell contents including microalgal oil are released out of the disrupted cell wall and cell membrane and exposed to form disrupted microalgae (step 14). The disrupted microalgae have all of the ingredients of microalgae including the microalgal oil and residues (i.e. non-oil ingredients) since there is not any artificially proceed for removing ingredient of disrupted microalgae being used thereon.

The disrupted microalgae is then directly mixed with a catalyst, an alcohol and a co-solvent (step 15) to perform a transesterification (step 16). That is, the disrupted microalgae is directly used as a raw material, or celled reacting material of the transesterification for providing the microalagl oil thereof to be reacted with the alcohol in the transesterification. After the transesterification, the microalgal oil in disrupted microalgae is transesterified into the fatty acid methyl esters (FAMEs), i.e. the biodiesel, and the disrupted microalgae forms a biodiesel mixture at least having the biodiesel and the residues.

In one embodiment, the volume of liquid, including the water, the alcohol and the co-solvent, of the disrupted microalgae can be adjusted prior to the transesterification for the optimization of the transesterification.

In one embodiment, the dry microalgae are mixed with the water, the alcohol and/or the co-solvent and then the disruption for the microalgae in this mixture is performed to obtain the disrupted microalgae. Besides, in one embodiment, the wet microalgae can also be mixed with the water, the alcohol and/or the co-solvent in advance and the disruption for the microalgae in this mixture is subsequently performed to obtain the disrupted microalgae. In these embodiments, the catalyst is added into the disrupted microalgae after the disruption process and disrupted microalgae having the catalyst can further be added thereinto the alcohol and/or the co-solvent.

The above-mentioned disruption process can be executed by biological way (e.g. via the enzyme), chemical way (e.g. via acid or base) and/or physical way (e.g. via microwave device/method or ultrasonicating device/method).

The above-mentioned catalyst is used to promote the transesterification of the microalgal oil and the alcohol, and can be, for example, an enzyme for promoting the transesterification, an acid catalyst, a base catalyst and a combination thereof. The enzyme for promoting the transesterification can be a lipase and immobilized on a supporter/particle to form an immobilized enzyme/lipase. The acid catalyst can be sulfuric acid (H2SO4), phosphoric acid (H3PO4), hydrochloric acid (HCl) or a combination thereof. The base catalyst can be a homogeneous one such as sodium hydroxide (NaOH), potassium hydroxide (KOH), sodium methoxide (NaOCH3), potassium methoxide (KOCH3), sodium amide (NaNH2), sodium hydride (NaH), potassium amide (KNH2), potassium hydride (KH) and a combination thereof, and a non-homogeneous one such as calcium oxide (CaO), magnesium oxide (MgO), strontium oxide (SrO) and a combination thereof. The homogeneous and the non-homogeneous base catalysts can also mixed for using in the present method.

The above-mentioned alcohol can be a short chain alcohol such as a methanol, an ethanol, a propanol, a butanol, a pentanol and a combination thereof. Besides, the co-solvent can be a hexane, an acetone, a chloroform, a petroleum ether and a combination thereof

TABLE 1 Group A Group B Group C Hexane Yield of Methanol Yield of Water Yield of (mL) biodiesel (%) (mL) biodiesel (%) (mL) biodiesel (%) 0 0 0 0 0 84.0 0.50 0 0.18 77.14 0.20 88.2 0.99 64.3 0.54 83.96 0.59 92.5 1.49 85.4 1.08 96.32 1.18 93.5 1.98 92.2 1.99 94.78 1.58 94.2 2.48 89.2 2.89 77.84 1.97 92.1 2.98 86.9 3.61 75.27 2.96 91.6 3.97 85.3 5.42 0 3.94 85.0 4.96 73.1 4.92 80.4 5.91 76.0

Table 1 shows the yield of biodiesel produced by dry microalgae containing 63.2% oil (w/w) via the method shown in FIG. 1. In Table 1, the methods for producing the biodiesel use immobilized lipase as the catalyst, and respective volumes of the hexane, the methanol and the water used in these methods are shown.

Specifically, the Group A shows the volume of hexane added into the dry microalgae (mL, per gram of the dry microalgae), in which 1.08 mL of the methanol per gram of the dry microalgae is added in each conditions (having different volume of hexane), and no water is further added in every conditions. The Group B shows the volume of methanol added into the dry microalgae (mL, per gram of the dry microalgae), in which 3.97 mL of the hexane per gram of the dry microalgae is added in each conditions (having different volume of methanol), and no water is further added in every conditions. The Group C shows the volume of water added into the dry microalgae (mL, per gram of the dry microalgae), in which 1.08 mL of the methanol and 3.97 mL of the hexane per gram of the dry microalgae are added in each conditions (having different volume of water). The dry microalgae in the mixture of hexane and methanol of all the conditions of Groups A, B and C are disrupted by ultrasonication, and 0.6 g of the immobilized lipase per gram of the dry microalgae is added in each the disrupted microalgae of all the conditions of Groups A, B and C. The reactive temperature, rotational speed of the rotation for mixing hexane, methanol and immobilized lipase (and water in Group C), and reactive duration of the mix of the hexane, methanol and immobilized lipase (and water in Group C) to perform the transesterification in all the conditions of Groups A, B and C are 40° C., 600 rpm and 48 hours respectively. The yield of biodiesel shown in Table 1 is calculated via dividing the total weight of the produced biodiesel at the termination (at the 48^(th) hour) of the transesterification reaction by the total weight of oil of the respective dry microalgae in all the conditions of Groups A, B and C.

As shown in Table 1, by using the dry microalgae as the raw material and adjusting the volumes of alcohol (methanol), co-solvent (hexane) or water mixed with the dry microalgae, the yield of biodiesel of the present method is higher than 90% and even up to 96%, which shows that the present method brings high yield of biodiesel.

TABLE 2 Methanol (mL) Yield of biodiesel (%) 0.22 92.15 0.36 94.88 0.72 71.35 1.08 66.02 1.44 50.32 1.81 40.81

Table 2 shows the yield of biodiesel produced by wet microalgae containing 60% to 80% water (w/w) and 63.2% oil (w/w, calculated and measured by drying the wet microalgae into dry ones) via the method shown in FIG. 1. In Table 2, the methods for producing the biodiesel use immobilized lipase as the catalyst, and respective volumes of the methanol used in these methods are shown.

Specifically, the Table 2 shows the volume of methanol added into the wet microalgae (mL, per gram of the wet microalgae), in which 0.79 mL of the hexane and 0.12 g of the immobilized lipase per gram of the wet microalgae is added in each conditions (having different volume of methanol). The wet microalgae in the mixture of hexane and methanol of all the conditions shown in Table 2 are disrupted by ultrasonication. The reactive temperature, rotational speed of the rotation for mixing hexane, methanol and immobilized lipase, and reactive duration of the mix of the hexane, methanol and immobilized lipase to perform the transesterification in all the conditions shown in Table 2 are 40° C., 600 rpm and 48 hours respectively.

As shown in Table 2, by using the wet microalgae as the raw material and adjusting the volumes of alcohol (methanol) mixed with the wet microalgae, the yield of biodiesel of the present method is up to 94%, which shows that the present method brings high yield of biodiesel.

TABLE 3 Content of Catalyst Hexane Methanol Yield of oil (wt %) (g) (mL) (mL) biodiesel (%) 13.5 1.03 1.37 0.69 90.09 15.2 0.82 1.14 0.58 92.12 29.8 0.33 0.91 0.46 92.22 45.6 0.18 0.68 0.35 93.34 63.2 0.11 0.46 0.23 96.63

Table 3 shows the yield of biodiesel produced by wet microalgae containing 86% to 91% water (w/w) via the method shown in FIG. 1. In Table 3, the methods for producing the biodiesel use immobilized lipase as the catalyst, and respective volumes of the methanol, the hexane and the immobilized lipase used in these methods are shown. Besides, the content of oil is calculated and measured by drying the respective wet microalgae into dry ones.

Specifically, the Table 3 shows the respective volumes of methanol, hexane and immobilized lipase per gram of the wet microalgae added into the wet microalgae. The wet microalgae in the mixture of hexane and methanol of all the conditions shown in Table 3 are disrupted by ultrasonication. The reactive temperature, rotational speed of the rotation for mixing hexane, methanol and immobilized lipase, and reactive duration of the mix of the hexane, methanol and immobilized lipase to perform the transesterification in all the conditions shown in Table 3 are 40° C., 600 rpm and 48 hours respectively.

As shown in Table 3, by adjusting the respective volumes of alcohol (methanol), co-solvent (hexane) or immobilized lipase mixed with the wet microalgae, the yields of biodiesel are all higher than 90% and even up to 96% despite the content of oil of wet microalgae being even down to 13.5%. Also, data in Table 3 reveal that the present method is suitable for the microalgae having various contents of oil.

TABLE 4 Dry microalgae Wet microalgae Water originally in 0 0 80 80 microalgae (wt %) Water additionally added 10 10 5 5 into microalgae (mL) Hexane (mL) 15 9 4 10 Methanol (mL) 5 3 4 3 Catalyst (g) 0.5 0.5 0.2 0.3 Yield of biodiesel (%) 74.9 85.6 73.4 96.0

Table 4 shows the yield of biodiesel produced by dry microalgae and wet microalgae containing 80% water (w/w) via the method shown in FIG. 1. In Table 4, the methods for producing the biodiesel use solid base catalyst of SrO having been modified as the catalyst, and contents of water and the respective volumes of the methanol, the hexane and the SrO used in these methods are shown. Besides, the respective contents of oil of dry microalgae and wet microalgae containing are 45.6%, wherein the oil content of wet microalgae is calculated and measured by drying the respective wet microalgae into dry ones.

Specifically, the Table 4 shows the respective volumes of additionally added water, methanol, hexane and SrO per gram of the dry or wet microalgae added into the dry or wet microalgae. The dry or wet microalgae mixed in those above-mentioned liquid of all the conditions shown in Table 4 are disrupted by microwave. The reactive temperature, rotational speed of the rotation for mixing hexane, methanol and SrO, and reactive duration of the mix of the hexane, methanol and SrO to perform the transesterification in all the conditions shown in Table 4 are 45° C., 600 rpm and 3 hours respectively.

As shown in Table 4, by using solid base catalyst of SrO as the catalyst and adjusting the respective volumes of alcohol (methanol), co-solvent (hexane), catalyst or water mixed with the dry or wet microalgae, the yield of biodiesel is even up to 96%.

The lipase used in all the conditions of Tables 1, 2 and 3 is extracted from Burkholderia sp. C20, and the lipase of Burkholderia sp. C20 is immobilized on a nano-scaled and alkyl-grafted particle of Fe₃O₄—SiO₂. The microalgae used in all the conditions of Tables 1 to 4 are microalga Chlorella vulgaris ESP-31. However, the lipase, the supporter/particle anchored by the lipase and the species of microalga can respectively be substituted by any appropriate one.

In one embodiment, the microalgae used in the present method for producing the biodiesel as described above are substituted by other biomasses having oil therein. Also, the disruption way, alcohol, co-solvent, catalyst and reactive conditions for the transesterification used in producing the biodiesel of which those other biomasses are used as the raw materials can respectively be substituted by or adjusted into any appropriate one.

EMBODIMENTS Embodiment 1

a method for producing a biodiesel, comprising steps of providing a microalga; disrupting the microalga to obtain a disrupted microalga; and mixing the disrupted microalga directly with a catalyst, an alcohol and a co-solvent to produce the biodiesel.

Embodiment 2 is a method as described in Embodiment 1, wherein the disrupted microalga has all of ingredients of the microalga, the all of ingredients include a microalgal oil and a residue, the microalgal oil and the alcohol are reacted in a transesterification, and the catalyst is used to promote the transesterification of the microalgal oil and the alcohol, and is one selected from the group consisting of a lipase, an acid catalyst, a base catalyst and a combination thereof.

Embodiment 3 is a method as described in Embodiment 2, wherein the catalyst is an immobilized lipase.

Embodiment 4 is a method as described in Embodiment 3, wherein the immobilized lipase includes a particle and a lipase of Burkholderia sp. C20 immobilized thereon.

Embodiment 5 is a biodiesel mixture having the biodiesel produced by the method as described in Embodiment 2 and the residue.

Embodiment 6 is a method as described in Embodiment 1, wherein the disrupted microalga is directly mixed with the catalyst, the alcohol and the co-solvent without removing any ingredient therefrom after the step of disrupting the microalga to obtain the disrupted microalga.

Embodiment 7 is a method as described in Embodiment 1, wherein the alcohol is a short chain alcohol being one selected from the group consisting of a methanol, an ethanol, a propanol, a butanol, a pentanol and a combination thereof, and the co-solvent is one selected from the group consisting of a hexane, an acetone, a chloroform, a petroleum ether and a combination thereof.

Embodiment 8 is a method as described in Embodiment 1, wherein the microalga has a species of Chlorella vulgaris ESP-31.

Embodiment 9 is a method as described in Embodiment 3, wherein the microalga is one selected from the group consisting of a dry microalga, a wet microalga and a combination thereof.

Embodiment 10: a method for producing a biodiesel, comprising steps of providing a microalga; disrupting the microalga to obtain a disrupted microalga; and performing a transesterification in which the disrupted microalga is directly used as a reacting material thereof to produce the biodiesel.

Embodiment 11 is a method as described in Embodiment 10 further comprising a step of directly mixing the disrupted microalga with a catalyst and an alcohol to perform the transesterification, wherein the disrupted microalga has whole ingredients of the microalga, the whole ingredients include a microalgal oil and a residue, the microalgal oil and the alcohol are reacted in the transesterification, and the catalyst is used to promote the transesterification of the microalgal oil and the alcohol.

Embodiment 12 is a method as described in Embodiment 11, wherein the disrupted microalga mixed with the catalyst and the alcohol is further mixed with a co-solvent for promoting the transesterification, the catalyst is one selected from the group consisting of a lipase, an acid catalyst, a base catalyst and a combination thereof, the alcohol is a short chain alcohol being one selected from the group consisting of a methanol, an ethanol, a propanol, a butanol, a pentanol and a combination thereof, and the co-solvent is one selected from the group consisting of a hexane, an acetone, a chloroform, a petroleum ether and a combination thereof.

Embodiment 13 is a method as described in Embodiment 11, wherein the catalyst is an immobilized lipase including a particle and a lipase of Burkholderia sp. C20 immobilized thereon.

Embodiment 14 is a biodiesel mixture having the biodiesel produced by the method as described in Embodiment 11 and the residue.

Embodiment 15: a method for producing a biodiesel, comprising steps of providing a biomass having an oil; obtaining a crude mixture from the biomass; and performing a transesterification in which the crude mixture is used as a reacting material thereof to produce the biodiesel.

Embodiment 16 is a method as described in Embodiment 15, wherein the crude mixture is obtained by disrupting the biomass, and the method further comprises a step of directly mixing the crude mixture with a catalyst and an alcohol without removing any ingredient from the crude mixture to perform the transesterification.

Embodiment 17 is a method as described in Embodiment 15 further comprising a step of directly mixing the crude mixture with a catalyst and an alcohol to perform the transesterification, wherein the crude mixture has whole ingredients of the biomass, the whole ingredients have an oil and a residue, the oil and the alcohol are reacted in the transesterification, and the catalyst is used to promote the transesterification of the oil and the alcohol.

Embodiment 18 is a method as described in Embodiment 17,

wherein the catalyst is an immobilized lipase including a particle and a lipase of Burkholderia sp. C20 immobilized thereon.

Embodiment 19 is a biodiesel mixture having the biodiesel produced by the method as claimed in claim 17 and the residue.

Embodiment 20 is a method as described in Embodiment 15,

wherein the biomass is a microalgae biomass having a species of Chlorella vulgaris ESP-31.

Based on the above embodiments, it is known, via the present method, if the biomass such as microalgae biomass are taken as the raw material for producing the biodiesel, the process of microalgal oil extraction can be omitted and the whole disrupted microalgae having the microalgal oil is directly used for the transesterification to produce the biodiesel. Accordingly, the present method for producing the biodiesel can not only use the whole microalgal oil of microalgae for the transesterification, but also save the cost of process of microalgal oil extraction. Furthermore, the present method can directly use the wet microalgae as the raw material for producing the biodiesel, which omits the process of drying the wet microalgae and saves the relevant cost thereof.

While the disclosure has been described in terms of what is presently considered to be the most practical and preferred embodiments, it is to be understood that the disclosure needs not be limited to the disclosed embodiments. Therefore, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims, which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures. 

What is claimed is:
 1. A method for producing a biodiesel, comprising steps of: providing a microalga; disrupting the microalga to obtain a disrupted microalga; and mixing the disrupted microalga directly with a catalyst, an alcohol and a co-solvent to produce the biodiesel.
 2. The method as claimed in claim 1, wherein the disrupted microalga has all of ingredients of the microalga, the all of ingredients include a microalgal oil and a residue, the microalgal oil and the alcohol are reacted in a transesterification, and the catalyst is used to promote the transesterification of the microalgal oil and the alcohol, and is one selected from the group consisting of a lipase, an acid catalyst, a base catalyst and a combination thereof.
 3. The method as claimed in claim 2, wherein the catalyst is an immobilized lipase.
 4. The method as claimed in claim 4, wherein the immobilized lipase includes a particle and a lipase of Burkholderia sp. C20 immobilized thereon.
 5. A biodiesel mixture having the biodiesel produced by the method as claimed in claim 2 and the residue.
 6. The method as claimed in claim 1, wherein the disrupted microalga is directly mixed with the catalyst, the alcohol and the co-solvent without removing any ingredient therefrom after the step of disrupting the microalga to obtain the disrupted microalga.
 7. The method as claimed in claim 1, wherein the alcohol is a short chain alcohol being one selected from the group consisting of a methanol, an ethanol, a propanol, a butanol, a pentanol and a combination thereof, and the co-solvent is one selected from the group consisting of a hexane, an acetone, a chloroform, a petroleum ether and a combination thereof.
 8. The method as claimed in claim 1, wherein the microalga has a species of Chlorella vulgaris ESP-31.
 9. The method as claimed in claim 1, wherein the microalga is one selected from the group consisting of a dry microalga, a wet microalga and a combination thereof.
 10. A method for producing a biodiesel, comprising steps of: providing a microalga; disrupting the microalga to obtain a disrupted microalga; and performing a transesterification in which the disrupted microalga is directly used as a reacting material thereof to produce the biodiesel.
 11. The method as claimed in claim 10 further comprising a step of directly mixing the disrupted microalga with a catalyst and an alcohol to perform the transesterification, wherein the disrupted microalga has whole ingredients of the microalga, the whole ingredients include a microalgal oil and a residue, the microalgal oil and the alcohol are reacted in the transesterification, and the catalyst is used to promote the transesterification of the microalgal oil and the alcohol.
 12. The method as claimed in claim 11, wherein the disrupted microalga mixed with the catalyst and the alcohol is further mixed with a co-solvent for promoting the transesterification, the catalyst is one selected from the group consisting of a lipase, an acid catalyst, a base catalyst and a combination thereof, the alcohol is a short chain alcohol being one selected from the group consisting of a methanol, an ethanol, a propanol, a butanol, a pentanol and a combination thereof, and the co-solvent is one selected from the group consisting of a hexane, an acetone, a chloroform, a petroleum ether and a combination thereof.
 13. The method as claimed in claim 11, wherein the catalyst is an immobilized lipase including a particle and a lipase of Burkholderia sp. C20 immobilized thereon.
 14. A biodiesel mixture having the biodiesel produced by the method as claimed in claim 11 and the residue.
 15. A method for producing a biodiesel, comprising steps of: providing a biomass having an oil; obtaining a crude mixture from the biomass; and performing a transesterification in which the crude mixture is used as a reacting material thereof to produce the biodiesel.
 16. The method as claimed in claim 15, wherein the crude mixture is obtained by disrupting the biomass, and the method further comprises a step of directly mixing the crude mixture with a catalyst and an alcohol without removing any ingredient from the crude mixture to perform the transesterification.
 17. The method as claimed in claim 15 further comprising a step of directly mixing the crude mixture with a catalyst and an alcohol to perform the transesterification, wherein the crude mixture has whole ingredients of the biomass, the whole ingredients have an oil and a residue, the oil and the alcohol are reacted in the transesterification, and the catalyst is used to promote the transesterification of the oil and the alcohol.
 18. The method as claimed in claim 17, wherein the catalyst is an immobilized lipase including a particle and a lipase of Burkholderia sp. C20 immobilized thereon.
 19. A biodiesel mixture having the biodiesel produced by the method as claimed in claim 17 and the residue.
 20. The method as claimed in claim 15, wherein the biomass is a microalgae biomass having a species of Chlorella vulgaris ESP-31. 